Tiling microarray analysis of rice chromosome 10 to identify the transcriptome and relate its expression to chromosomal architecture

Abstract

Background: Sequencing and annotation of the genome of rice (Oryza sativa) have generated gene models in numbers that top all other fully sequenced species, with many lacking recognizable sequence homology to known genes. Experimental evaluation of these gene models and identification of new models will facilitate rice genome annotation and the application of this knowledge to other more complex cereal genomes.

Results: We report here an analysis of the chromosome 10 transcriptome of the two major rice subspecies, japonica and indica, using oligonucleotide tiling microarrays. This analysis detected expression of approximately three-quarters of the gene models without previous experimental evidence in both subspecies. Cloning and sequence analysis of the previously unsupported models suggests that the predicted gene structure of nearly half of those models needs improvement. Coupled with comparative gene model mapping, the tiling microarray analysis identified 549 new models for the japonica chromosome, representing an 18% increase in the annotated protein-coding capacity. Furthermore, an asymmetric distribution of genome elements along the chromosome was found that coincides with the cytological definition of the heterochromatin and euchromatin domains. The heterochromatin domain appears to associate with distinct chromosome level transcriptional activities under normal and stress conditions.

Conclusion: These results demonstrated the utility of genome tiling microarray in evaluating annotated rice gene models and in identifying novel transcriptional units. The tiling microarray sanalysis further revealed a chromosome-wide transcription pattern that suggests a role for transposable element-enriched heterochromatin in shaping global transcription in response to environmental changes in rice.

Figure 1

Processing the rice chromosome 10 tiling microarray hybridization data. (a) Distribution of fluorescence intensity of all positive and negative features of the four indica N Arrays. (b) All eight distributions were scaled to have a uniform intensity peak value at 8 (log₂). (c) Mathematic model for determination of signal probes. A bimodal distribution of log₂ background-adjusted intensity of all positive features is used to model the noise as a normal distribution by mirroring the distribution of low intensity (< 6 of log₂). A cutoff value corresponding to a 90% confidence level to reject noise probes according to the modeled noise distribution is indicated. (d) Distribution of hybridization rate in the exonic and intronic regions of rice chromosome 10. Hybridization rate (HR) is calculated as the ratio of the number of signal probes against the total number of interrogating probes per kilobase of sequence.

Figure 2

Tiling microarray analysis of the rice chromosome 10 transcriptome. (a) Schematic representation of rice chromosome 10. The purple oval denotes the centromere. (b) A region from the long arm of chromosome 10 displaying the three sets of gene models used: BGI indica; TIGR japonica and BGI japonica. The nonredundant protein-coding gene models are aligned to the chromosomal sequences and color-coded on the basis of their classification (see text). (c) Detailed tiling profile of one representative CG model. The model is represented here as block arrows, which point in the direction of transcription. Signal oligos are aligned according to their chromosomal coordinates. The fluorescence intensity value of each signal oligo, capped at 2,500, is depicted as a vertical bar. The shade of the bar represents the oligo index score (see Materials and methods). The red blocks underneath the bars indicate the presence of an interrogating oligo in the microarray.

Figure 3

Cloning and sequence analysis of japonica chromosome 10 UG models and intergenic TARs. (a) Summary of RT-PCR analysis of selected UG models. ORF identical, annotated ORF is the same as determined from the cloned sequence; ORF different, annotated ORF is different from that in the cloned sequence. (b) Summary of RT-PCR analysis of selected intergenic TARs. Gene model, cloned TARs overlapping with TIGR models; BGF prediction, cloned TARs overlapping with BGF predictions; unique, cloned TARs not overlapping with any annotated feature. (c) Representative UG models whose cloned sequences either differ from (OsJN02936) or are the same as (OsJN03072) the annotated ones. (d) Representative intergenic TARs whose cloned sequences either overlap with a TIGR model (OsJN01855) or are completely intergenic (C10_ZN376). Representation of microarray data in this figure is the same as in Figure 2 except that the oligo index is omitted.

Figure 4

Analysis of intergenic TARs of japonica chromosome 10. (a) The 988 japonica chromosome 10 intergenic TARs distributed by length. (b) RNA gel blotting analysis of selected japonica intergenic TARs. Probes for the intergenic TARs shown in this panel were derived from corresponding PCR-amplified TAR sequences from japonica rice genomic DNA. (c) Probes shown in this panel were derived from RT-PCR amplification of the corresponding TARs from poly(A)⁺ RNA. (d) The rice cDNAs for eIF4A and actin2 were used as loading controls. 5 μg of RNA from the four sources - root, shoot, panicle, and suspension cell culture - that were used for probing tiling microarrays were used for RNA blot analysis here.

Figure 5

Comparison and integration of chromosome 10 gene models. (a) Number of annotated and array-detected high homology (HH) and low homology (LH) models in the BGI indica, BGI japonica, and TIGR japonica annotations. (b) The 549 new gene models were combined with the 3,019 TIGR models. Origins of the new models are shown on the left. Expression support for the TIGR models is shown on the right. Expressed, models matching full-length cDNA/EST; array-detected, models not supported by the expressed sequences but detected by microarray; undetected, models neither supported by expressed sequences nor detected by microarray. (c) Classification of integrated japonica chromosome 10 gene models based on tiling array detection and exon number (left), homology to Arabidopsis genes (middle), and previous expression or homology support to the models (right).

Figure 6

Rice chromosome 10 gene model distribution and expression. (a) Characterization of TIGR nonredundant protein-coding gene models. Model density, array detection rate, number of signal oligos, number of intergenic TARs, and cumulative length (in kilobases) of masked oligos are calculated in 100-kb windows along the length of chromosome 10, and are represented by color-coded vertical bars. A scale representing the physical length of chromosome 10 is shown at the bottom of the panel. The arrowhead delimits the division of domain I and domain II as indicated in the text. Note that the centromere is located at a position around 7 to 8 Mb in chromosome 10. (b) Gene model density and array detection rate of the BGI japonica annotation. (c) Gene model density and array detection rate of the BGI indica annotation. (d) Comparison of the S Arrays and the N Arrays using the BGI japonica annotation. Log₂ (S/N) of the hybridization intensity was calculated for individual models (top) and the mean intensity of all models in 100-kb windows along the length of chromosome 10 (bottom).

Figure 7

Chromosome-wide distribution of gene models and chromosomal elements. (a) Distribution of TIGR japonica nonredundant protein-coding gene models (non-TE) and transposable element-related models (TE) in 1-Mb windows across chromosome 10. The division between domain I and II is indicated by the arrowhead. Note that the centromere is located at around 7 to 8 Mb in chromosome 10. (b) Distribution of BGI japonica CG and UG models in 1-Mb windows across chromosome 10. (c) Distribution of BGI japonica HH and LH models in 1-Mb windows across chromosome 10. (d) Numbers of the TIGR japonica nonredundant protein-coding gene models (TIGR Non-TE) and tiling array-detected intergenic TARs in 1-Mb windows across chromosome 10.

Figure 8

Colinearity of the CG models for chromosome 10 in japonica and indica rice. (a) Chromosomal positions of corresponding CG model pairs along chromosome 10 in japonica (blue) and indica (red) rice are plotted against the sequential orders of the CG pairs. (b) Physical distance between corresponding CG pairs is plotted against their sequential orders along the chromosome.